類蕈孢素氨基酸是僅由淡海水藻類產生的光保護化合物，為一群能保護生物不被太陽輻射傷害的二次代謝物。利用高效液相色層分析儀偵測七種海洋微細藻，結果顯示渦鞭藻屬的小亞歷山大藻 ( Alexandrium minutum ) 會生成類蕈孢素氨基酸，且 AM-2、AM-3、AM-4、AM-6 株擁有而 AM-5 則否。探討溫度及光線對 AM-3 藻粉萃取的類蕈孢素氨基酸之穩定性，顯示高溫 ( 50℃ ) 在長時間 ( 35 days ) 會造成較大破壞，而紫外線會造成其在 1 天後減少 48 %、第 2 天便消失。分析小亞歷山大藻在不同鹽度、營養鹽及紫外線照射下，細胞之增殖與類蕈孢素氨基酸之產生，以瞭解這些因子間的關係。當鹽度上升 ( 15 psu、25 psu、35 psu、45 psu ) 小亞歷山大藻細胞密度顯著下降，但各組間類蕈孢素氨基酸產量並無不同。當提供更多的氮營養鹽 ( 2 N ) 時，在較低氮營養鹽濃度組 ( N / 10、N / 2 ) 藻體產生更多的類蕈孢素氨基酸。缺乏氮營養鹽同時給予紫外線的實驗組，其類蕈孢素氨基酸量下降，並無誘發其產生。而足夠氮營養鹽 ( 2 N ) 的情況下，紫外線能誘發類蕈孢素氨基酸的產生。實驗結果顯示，類蕈孢素氨基酸可能為一種細胞內儲存氮元素的方式，此物質並不肩負著調節滲透壓的功能。紫外線為小亞歷山大藻誘發類蕈孢素氨基酸的原因。

The photoprotect compound mycosporine-like amino acids ( MAAs ) are a family of secondary metabolites can protect organisms exposed to solar ultraviolet radiation ( UVR ), only produced in the algae. Analyzed by HPLC, only Alexandrium minutum Halim ( Dinophyceae ) produces MAAs among seven marine microalgae species. The strains named AM-2、AM-3、AM-4 and AM-6 showed the positive results for producing the MAAs, but not AM-5. Researches on the stability the MAAs extracts from algal powder ( AM-3 ) hold at 4℃, 25℃ and 50℃ for 35 days and light expose. The results showed the MAAs gradually decayed after 35 days at 50℃, after exposed to UVR but decreased 48 % in one day and disappeared in two days. The growth and the MAAs produced in A. minutum ( AM-4, AM-6 ) under different salinity, nitrate concentration and UVR were analyzed to find their relationships. When the salinity increased ( 15 psu、25 psu、35 psu、45 psu ), the cell densities of both strain decreased significantly, but with similar MAAs composition and contents. In higher nitrate nutrient ( 2 N ), the cells produced more MAAs than those cultivated in lower nitrate ( N / 10、N / 2 ). Under the UV treatment with the deficient nitrate nutrient, the concentration of MAAs in A. minutum decreased after exposed to UVR for 14 hours. However, provided enough nitrate nutrient ( 2 N ), UVR can induced A. minutum to produce higher concentration of MAAs. These results showed MAAs may serve as a nitrogenous compound for intracellular storage, and not as a substance for osmotic adjustment. UVR is an important factor which induce MAAs produced in A. minutum.

目錄

謝辭................................................................I
中文摘要...........................................................II
英文摘要...........................................................III
目錄...............................................................IV
表目錄............................................................VII
圖目錄...........................................................VIII
附錄目錄...........................................................X
壹、前言............................................................1
一、藻華發生之歷史、原因及危害........................................1
二、藻毒種類及對人類危害機制.........................................1
三、小亞歷山大藻.....................................................2
(一) 在台灣產生的毒害事件及環境.....................................2
(二) 發生紀錄及生活史...............................................3
(三) 型態分類與分生分類.............................................3
四、防曬方法與物質...................................................4
(一) 紫外線光譜.....................................................4
(二) 臭氧破洞及其造成影響.....................................5
(三) 紫外線對人類皮膚之傷害.....................................5
(四) 生物抗紫外線之物質與方法.....................................6
五、類蕈孢素氨基酸...................................................7
(一) 發現過程.......................................................7
(二) 化學構造及在生物體內代謝途徑...................................8
(三) 藻類含類蕈孢素氨基酸之研究.....................................9
(四) 光與環境對類蕈孢素氨基酸的影響.................................9
六、研究動機........................................................10
貳、材料與方法......................................................11
一、類蕈孢素氨基酸的分析與微細藻種類篩選............................11
(一) 凍存藻粉篩選..................................................11
(二) 類蕈孢素氨基酸分析............................................11
1. 類蕈孢素氨基酸的萃取........................................11
2. 高效液相色層分析儀系統......................................11
(a) 儀器設備................................................11
(b) 沖提液..................................................12
(c) 操作步驟................................................12
3. 類蕈孢素氨基酸之收集與其吸收波長測定........................13
二、 類蕈孢素氨基酸對溫、光的敏感性..................................13
三、 小亞歷山大藻的培養操作.........................................13
(一) 培養液配製....................................................14
1. f / 2 營養鹽..................................................14
2. Walne 營養鹽..................................................14
(二) 養殖用水及器皿處理............................................15
1. 海水........................................................15
2. 培養容器....................................................15
3. 打氣培養....................................................15
(三) 培養操作......................................................15
1. 種原維持....................................................15
2. 靜置培養....................................................16
3. 1 L 至 10 L 打氣培養........................................16
四、不同因子對小亞歷山大藻產類蕈孢素氨基酸的影響....................17
(一) 藻粉製成......................................................17
(二) 鹽度影響......................................................17
(三) 氮營養鹽改變..................................................17
(四) 紫外線照射....................................................17
(五) 增殖測定......................................................18
1. 藻細胞收集及計數...........................................18
2. 藻細胞增殖率...............................................18
(六) 水中硝酸氮 ( NO3 – N ) 營養鹽分析...............................18
1. 試劑........................................................19
2. 檢量線製備..................................................19
3. 測定實驗步驟................................................19
参、 結果...........................................................20
一、產類蕈孢素氨基酸藻種的篩選......................................20
二、類蕈孢素氨基酸的 HPLC 圖譜.....................................20
三、類蕈孢素氨基酸對光、溫的敏感性...................................20
四、鹽度改變對小亞歷山大藻產類蕈孢素氨基酸的影響....................21
五、硝酸鹽改變對小亞歷山大藻產類蕈孢素氨基酸的影響..................22
六、 紫外線照射對小亞歷山大藻產類蕈孢素氨基酸的影響.................23
肆、討論............................................................24
伍、參考文獻........................................................33
陸、表..............................................................53
柒、圖..............................................................59
捌、附錄............................................................75

表目錄

表一、凍存藻粉之類蕈孢素氨基酸篩選..................................53
表二、f / 2 營養鹽配方...............................................54
表三、Walne營養鹽配方..............................................55
表四、單位重量不同處理在不同天數後類蕈孢素氨基酸以 HPLC 測定的波峰相對高度.............................................................56
表五、在硝酸鹽試驗中小亞歷山大藻的增殖率............................57
表六、單位重量下小亞歷山大藻以不同氮營養鹽培養後其萃取的類蕈孢素氨基酸在 HPLC 圖譜上波峰的相對高度......................................58

圖目錄

圖1. 小亞歷山大藻甲醇萃取液之吸光圖譜..............................59
圖2. 小亞歷山大藻水萃取類蕈孢素氨基酸之 HPLC 圖...................59
圖3. 小亞歷山大藻水萃取類蕈孢素氨基酸之 HPLC 圖譜 ( 圖 2 ) 波峰 1 純化後之吸光圖譜.....................................................60
圖4. 小亞歷山大藻水萃取類蕈孢素氨基酸之 HPLC 圖譜 ( 圖 2 ) 波峰 2 純化後之吸光圖譜.....................................................60
圖5. 小亞歷山大藻水萃取類蕈孢素氨基酸之 HPLC 圖譜 ( 圖 2 ) 波峰 3 純化後之吸光圖譜.....................................................61
圖6. 小亞歷山大藻水萃取類蕈孢素氨基酸之 HPLC 圖譜 ( 圖 2 ) 波峰 4 純化後之吸光圖譜.....................................................61
圖7. 藻粉萃取之類蕈孢素氨基酸在 4℃ 無光照下第 0 天、第 7 天、第 14 天、第 21 天、第 35 天、第 49 天及 HPLC 圖譜.........................62
圖8. 藻粉萃取之類蕈孢素氨基酸在 25℃ 無光照下第 0 天、第 35 天之 HPLC圖譜...............................................................62
圖9. 藻粉萃取之類蕈孢素氨基酸在 25℃ 有光照下第 0 天、第 35 天之 HPLC圖譜...............................................................63
圖10. 藻粉萃取之類蕈孢素氨基酸在 50℃ 無光照下第 0 天、第 14 天、第 35 天、第 42 天之 HPLC 圖譜...........................................63
圖11.藻粉萃取之類蕈孢素氨基酸水萃取物經紫外線照射第 0 天、第 1 天之 HPLC 圖譜.........................................................64
圖12.藻粉萃取之類蕈孢素氨基酸甲醇萃取物經紫外線照射第 0 天、第 1 天、第 2 天、第 3 天之 HPLC 圖譜.......................................64
圖13.小亞歷山大藻 AM-6 及 AM-4 從 15 psu 移植至不同鹽度養殖 6 天的細胞數.............................................................65
圖 14. 小亞歷山大藻 AM-6、AM-4 在鹽度試驗下其培養液中硝酸鹽濃度...66
圖15.小亞歷山大藻 AM-6 在鹽度試驗下其類蕈孢素氨基酸之 HPLC 圖譜.67
圖16.小亞歷山大藻 AM-4 在鹽度試驗下其類蕈孢素氨基酸之 HPLC 圖譜.67
圖17.小亞歷山大藻 AM-6 在不同硝酸鹽濃度下之生長情形..............68
圖18.小亞歷山大藻 AM-4 在不同硝酸鹽濃度下之生長情形..............68
圖19.小亞歷山大藻 AM-6 在不同硝酸鹽濃度培養 7 天或 11 天後培養液中硝酸鹽濃度.........................................................65
圖20.小亞歷山大藻 AM-4 在不同硝酸鹽濃度培養 7 天後培養液中硝酸鹽濃度.................................................................69
圖21.小亞歷山大藻 AM-6 在不同氮營養鹽後其類蕈孢素氨基酸萃取之 HPLC 圖譜...............................................................70
圖22.小亞歷山大藻 AM-4 在不同氮營養鹽濃度培養下 7 天後其類蕈孢素氨基酸之 HPLC 圖譜....................................................71
圖23.小亞歷山大藻 ( AM-6 ) 培養在硝酸鹽1 N 下 6 天後照射紫外線 1 天後其類蕈孢素氨基酸之 HPLC 圖譜......................................72
圖24.小亞歷山大藻 ( AM-4 ) 培養在硝酸鹽 1 N 下 6 天後照射紫外線 1 天後其類蕈孢素氨基酸之 HPLC 圖譜....................................72
圖25.小亞歷山大藻 ( AM-4 ) 氮充足下空白組與照射紫外線組其類蕈孢素氨基酸之 HPLC 圖譜....................................................73
圖26.小亞歷山大藻 ( AM-4 ) 氮充足空白組與照射紫外線組其類蕈孢素氨基酸之吸光圖譜 ( 實線為紫外線照射處理、虛線為對照組 )...................74

附錄目錄

附錄一、附錄一、蕈孢素及類蕈孢素氨基酸結構...........................75
附錄二、經莽草酸代謝生成類蕈孢素胺基酸之路徑........................76
附錄三、類蕈孢素胺基酸代謝途徑......................................77

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